PROJECT SUMMARY Nucleosomes are not only the building blocks of eukaryotic chromatin, but also major carriers of epigenetic information. Shortly following DNA replication, new nucleosomes are assembled on the nascent strands from a mixture of recycled parental histones and newly synthesized ones. This process allows the chromatin state to be faithfully transmitted for many generations, while also enabling the offspring to reset its epigenome if necessary. Another principal, somewhat less appreciated, characteristic of chromatin replication is that it is inherently asymmetric, with the leading and lagging strands synthesized by drastically different machineries and pathways. Such asymmetry entails that each of the two daughter strands incorporates a distinct set of histones, thereby capable of adopting different chromatin states. Asymmetric histone segregation plays a pivotal role in cell fate decisions during development and stem cell maintenance. However, whether histones are deterministically or randomly distributed on nascent DNA, and how the distribution pattern depends on chromatin contexts, remain poorly understood. This is largely due to a lack of experimental tools with which to distinguish between leading- and lagging-strand deposition of histones. Using budding yeast as a model organism, this project aims to develop novel methodologies to probe histone inheritance in a strand-specific manner. At the molecular level, the chain of events during replication-coupled chromatin assembly will be directly visualized by correlative single-molecule fluorescence-optical tweezers microscopy. The leading and lagging strands will be spatially separated via microfluidic control so that the destination of deposited histones can be unambiguously assigned. This assay will then be used to dissect the functions of histone chaperones in mediating nucleosome disruption and reassembly. At the cellular level, a cell-cycle-dependent pulse-labeling strategy will be adopted to distinguish newly synthesized histones from preexisting ones. Histone distribution at the replication fork will be imaged and cell-to-cell variability assessed by super-resolution microscopy. At the systems level, nucleosomes on the leading or lagging strands will be selectively isolated through the usage of yeast strains carrying corresponding error-prone replicative polymerase variants. The genomic positions of these strand-selective nucleosomes will be mapped by next-generation sequencing and their chemical compositions analyzed by quantitative mass spectrometry. Overall, this multi-pronged approach will establish a framework for characterizing the differential partitioning of histones, their variants and post-translational modifications behind the replication fork, and for understanding the role of asymmetry in driving epigenome evolution and creating heterogeneity of cell identity. This project also paves the way for investigating how the dynamics of epigenetic inheritance alter in development and disease, thereby providing new therapeutic targets and strategies for developmental disorders, ageing, and cancer.